Efficient production of cembratriene-ol in Escherichia coli via systematic optimization

Background The tobacco leaf–derived cembratriene-ol exhibits anti-insect effects, but its content in plants is scarce. Cembratriene-ol is difficult and inefficiently chemically synthesised due to its complex structure. Moreover, the titer of reported recombinant hosts producing cembratriene-ol was low and cannot be applied to industrial production. Results In this study, Pantoea ananatis geranylgeranyl diphosphate synthase (CrtE) and Nicotiana tabacum cembratriene-ol synthase (CBTS) were heterologously expressed to synthsize the cembratriene-ol in Escherichia coli. Overexpression of cbts*, the 1-deoxy-d-xylulose 5-phosphate synthase gene dxs, and isopentenyl diphosphate isomerase gene idi promoted the production of cembratriene-ol. The cembratriene-ol titer was 1.53-folds higher than that of E. coli Z17 due to the systematic regulation of ggpps, cbts*, dxs, and idi expression. The production of cembratriene-ol was boosted via the overexpression of genes ispA, ispD, and ispF. The production level of cembratriene-ol in the optimal medium at 72 h was 8.55-folds higher than that before fermentation optimisation. The cembratriene-ol titer in the 15-L fermenter reached 371.2 mg L− 1, which was the highest titer reported. Conclusion In this study, the production of cembratriene-ol in E. coli was significantly enhanced via systematic optimization. It was suggested that the recombinant E. coli producing cembratriene-ol constructed in this study has potential for industrial production and applications. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-023-02022-4.


Background
Natural products include terpenoids, polyketides, phenylpropanoids, alkaloids, and so on [1]. Terpenoids and their derivatives are compounds obtained from mevalonic acid with the isoprene unit (C5) as the basic structural unit in the molecular skeleton [2]. Due to their unique and diverse bioactive characteristics, terpenoids play an important role in fields and applications such as pharmaceuticals, spices, food, biofuels, cosmetics, and industrial raw materials [3][4][5][6], which have attracted the interest of many researchers. Cembratriene-ol is a monocyclic diterpenoid that exhibits good insect deterrent effects [7][8][9][10][11]. This compound is a plant natural product that is mainly found in Nicotiana plants [7], and it can be completely degraded in the natural environment without residue, making it a sustainable biopesticide ingredient [12,13]. The titer of natural products extracted from plants cannot commonly meet the demands of their industrial application due to the lack of efficient production methods [1,14]. The content of cembratriene-ol in plants is low (0.18% of the leaf dry weight) [10,15], and direct extraction of cembratriene-ol from plants is difficult and expensive [15]. Moreover, cembratriene-ol is difficult and inefficiently chemically synthesised due to its complex structure [16]. The total chemical synthesis of plant natural products commonly produces significant waste, which may lead to further pollution [17].
Heterologous production pathways of natural chemicals in their natural production hosts can be transplanted or employed to construct engineered microbial cells producing these natural chemicals [1]. The biosynthesis of plant natural products with high value by engineered microbial cell factories has great prospects for their industrial production and application and provides a prospective solution for protecting plants and land and averting pollution from chemical synthesis [14,18,19]. Many studies have reported on heterologous synthesis of plant-based products in engineered microbial cell factories, such as cannabinoids [20], isoprenoids [21], and glycyrrhetinic acid [22]. Genetic and metabolic engineering methods have been used to optimize and modify metabolic pathways of microbial cells to efficiently produce high value chemicals [23]. Sophisticated metabolic engineering approaches can be employed to redirect the metabolic flux of microbial cells toward the formation of target products [1]. Moreover, systems metabolic engineering methods can be performed to construct an efficient microbial cell factory by generating elucidating metabolic processes and integrating these techniques with genetic engineering, synthetic biology, and systems biology [24][25][26][27].

Results and discussion
Construction of recombinant E. coli producing cembratriene-ol E. coli contains the natural DXP pathway, which can synthesise terpenoid skeleton chemicals. This pathway includes seven enzyme-catalysed reaction steps and requires NADPH and ATP [1]. Farnesyl pyrophosphate (FPP) can be synthesised and accumulated via the natural DXP pathway in E. coli. Based on the initial product FPP, cembratriene-ol can be produced via heterologous expression of geranylgeranyl pyrophosphate synthase and cembratriene-ol synthase in E. coli [12]. In this study, the geranylgeranyl pyrophosphate synthase gene ggpps and cembratriene-ol synthase gene cbts were heterologously expressed to synthesize cembratriene-ol in E. coli (Additional file 1: Fig. S1/S2/S3a). As shown in Fig. 2a, recombinant E. coli Z14 was constructed, with a titer of 1.58 × 10 − 2 mg L − 1 . The control strain without ggpps and cbts genes could not produce cembratriene-ol. Genes ggpps and cbts were expressed in E. coli, and cembratriene-ol was successfully synthesised. The productivity and conversion yield of glucose to cembratriene-ol in Z14 were 3.3 × 10 − 4 mg L − 1 ·h − 1 and 0.04 mg g − 1 at 48 h, respectively (Fig. 2b, Additional file 1: Fig. S4). Since the growth rate is one of significant factors for the specific productivity [33], the cell growth and growth rate were determined in this study. The maximum cell density (OD 600 ) and specific growth rate of Z14 were 3.73 and 0.24 h − 1 , which of the control was 3.65 and 0.27 h − 1 , respectively (Additional file 1: Fig. S3b/c). In the previous study, the cembratriene-ol toxicity effects were not observed for E. coli at the experimental concentration (0.25%) [12].

Effect of the cembratriene-ol synthase and its truncated variant on cembratriene-ol production
As a key enzyme in the synthesis of cembratriene-ol, cembratriene-ol synthase is of great significance for the production of cembratriene-ol in recombinant E. coli. Mischko et al. expressed one cembratriene-ol synthase lacking the plastid transit peptide to synthesize cembratriene-ol [12]. In this study, the effects of the cembratriene-ol synthase gene cbts and its truncated variant cbts* on the synthesis of cembratriene-ol in recombinant E. coli were compared, and the recombinant E. coli Z14 and E. coli Z15 were constructed (Additional file 1: Fig.  S1/S5a). The cembratriene-ol titer and productivity in E. coli Z15 expressing cbts* were increased by 20-folds higher than those of E. coli Z14 (Fig. 2a). The cbts* gene was also found to be more conducive to synthesizing cembratriene-ol in recombinant E. coli than cbts. It was found that the solubility of enzyme CBTS with plastid transit peptide sequence was reduced compared with CBTS* when they were heterologously overexpressed in E. coli (Additional file 1: Fig. S6). The conversion yield of glucose to cembratriene-ol in E. coli Z15 was 2-folds higher than that of E. coli Z14, indicating that the overexpression of cbts* facilitates the conversion of glucose to cembratriene-ol. The growth of E. coli Z15 was inhibited by the expression of cbts*, and the main reason may be that the large amount of glucose was used for synthesis of cembratriene-ol instead of cell growth (Additional file 1: Fig. S5b/c).

Effect of overexpressing key genes dxs and idi in carbon metabolism flux on cembratriene-ol production
The supply of isoprene pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) is very important for the production of cembratriene-ol [12]. The 1-deoxy-d-xylulose 5-phosphate synthase DXS (product of the dxs gene) and IPP isomerase IDI (product of the idi gene) are two key rate-controlling enzymes in the DXP pathway of E. coli. The gene idi plays a significant role in controlling the isoprenoid flux [33]. Park et al. overexpressed IDI and geranylgeranyl diphosphate synthase GPS from Archaeoglobus fulgidus to produce astaxanthin in an engineered E. coli [34]. Genes dxs and idi were coexpressed using plasmid pACYCDuet-1 in E. coli Z15 to construct recombinant E. coli Z16 (Additional file 1: Fig. S1/S7a). The titer (1.19 mg L − 1 ) of cembratriene-ol in E. coli Z16 was increased by 3.75-folds that of E. coli Z15 (Fig. 2a). The productivity of cembratriene-ol in E. coli Z16 was increased from 6.6 × 10 − 3 mg L − 1 ·h − 1 in E. coli Z15 to 2.5 × 10 − 2 mg L − 1 ·h − 1 , which indicated that overexpression of dxs and idi increased the carbon metabolism flux of the DXP pathway to cembratriene-ol and further enhanced the production of cembratriene-ol in recombinant E. coli. It was also reported that genes dxs and idi were overexpressed to improve the production of heterologous terpene (e.g. cembratriene-ol) [12,35]. The growth ability of E. coli Z16 was inhibited to a certain extent compared with E. coli Z15, suggesting that overexpression of dxs and idi increased the flux of carbon metabolism to cembratriene-ol but not cell growth (Additional file 1: Fig. S7b/c). E. coli Z16 had a higher residual glucose concentration (1.27 g L − 1 ) in fermentation liquid compared with that of E. coli Z15, and the conversion yield of glucose to cembratriene-ol was 5.5folds that of E. coli Z15 (Fig. 2b). Overexpression of dxs and idi was further verified to improve the conversion efficiency of glucose to cembratriene-ol via the DXP pathway in E. coli.
To further enhance the production of cembratriene-ol, plasmid pCDFDuet-1 was used to increase the expression levels of idi and dxs, and the recombinant E. coli Z17 was constructed (Additional file 1: Fig. S1/S8a). The titer and productivity of cembratriene-ol of E. coli Z17 reached 3.03 mg L − 1 and 6.3 × 10 − 2 mg L − 1 ·h − 1 , respectively, which was increased by 2.6-folds that of E. coli Z16 (Fig. 2). It was suggested that enhancing the expression levels of idi and dxs was beneficial for the efficient production of cembratriene-ol by recombinant E. coli. Yuan et al. also found a medium-copy plasmid pDCQ108 was used to express key enzymes (e.g., idi and dxs) to result in a significant increase in production of β-carotene compared with that of the low-copy plasmid pPCB15 [33]. Cell growth of E. coli Z17 was inhibited to a certain extent compared with E. coli Z16 (Additional file 1: Fig. S8b/c), and the carbon metabolism flux in the DXP pathway to cembratriene-ol should be one of the main reasons. The residual glucose concentration in the fermentation liquid of E. coli Z17 was close to 0, and the conversion yield of glucose to cembratriene-ol reached 0.763 mg g − 1 , which was 1.75-folds that of E. coli Z16. The enhanced expression levels of idi and dxs were beneficial for converting glucose to cembratriene-ol. Plasmid pRSFDuet-1 with a high copy number was used to increase the expression levels of ggpps and cbts* in E. coli Z17 for efficient synthesis of cembratriene-ol, and recombinant E. coli Z18 was constructed (Additional file 1: Fig. S8a). The titer and productivity of cembratriene-ol in E. coli Z18 was not significantly improved, and it was indicated that overexpression of ggpps and cbts* under theseconditions could not yield high efficiency cembratriene-ol production.

Enhancing cembratriene-ol production via systematic regulation of key gene expression
The DXP pathway contains multiple complex regulatory elements that are difficult to systematically optimise to enhance precursor accumulation (e.g. FPP). The accumulation of sufficient precursors plays an important role in producing the target compounds. Enhancing the biosynthetic flux of precursors in the DXP pathway in E. coli is a key strategy for increasing the accumulation of target products [1]. Based on the above results, it was found that the different expression levels of key genes idi, dxs, ggpps, and cbts* have an important influence on cembratriene-ol production in E. coli. The plasmids pACYCDuet-1, pCDFDuet-1, pETDuet-1, and pRSFDuet-1 were used to regulate the expression levels of key genes idi, dxs, ggpps, and cbts*, and four recombinant E. coli Z19, E. coli Z20, E. coli Z21, and E. coli Z22 were constructed (Additional file 1: Fig. S1/S9a). The theoretical copy numbers of plasmids pACYCDuet-1, pCDFDuet-1, pETDuet-1, and pRSFDuet-1 are 10, 20, 40, and 100, respectively [36]. In this study, the actual copy numbers of empty plasmids pACYCDuet-1, pCD-FDuet-1, pETDuet-1, and pRSFDuet-1 are 3.38, 4.89, 9.12, and 26.3 at the cultivation conditions, respectively (Additional file 1: Fig. S10). The titers of cembratriene-ol in E. coli Z19, E. coli Z20, E. coli Z21, and E. coli Z22 were 2.80, 4.63, 2.89, and 2.35 mg L − 1 , respectively (Fig. 2a). The titer of cembratriene-ol in E. coli Z20 was the highest, exhibiting an increase by 1.53-folds compared with that of E. coli Z17. The productivity of cembratriene-ol in E. coli Z20 was 9.6 × 10 − 2 mg L − 1 ·h − 1 , which was significantly higher than that of E. coli Z17. E. coli Z20 had the highest conversion yield of glucose to cembratrieneol (1.32 mg g − 1 ). It was presumed that this key enzyme expression mode (pCDFDuet-1 with idi and dxs, pRSF-Duet-1 with cbts*, and pETDuet-1 with ggpps) is beneficial for converting glucose to cembratriene-ol. The systemic regulation of idi, dxs, ggpps, and cbts* expression levels inhibited cell growth compared to that of the control (Additional file 1: Fig. S9b/c). The order of gene expression levels for improving the synthesis ability of cembratriene-ol in E. coli was cbts* > ggpps > idi > dxs, suggesting that overexpression of cbts* and ggpps using plasmids with high copies was preferred for the efficient synthesis of cembratriene-ol. These results indicated that cbts* and ggpps were critical genes for the efficient synthesis of cembratriene-ol in E. coli. Wang et al. also found that geranylgeranyl diphosphate synthase was a major rate-limiting enzyme for the biosynthesis of isoprenoids in engineered E. coli [34,36].
Enhancing cembratriene-ol production by overexpressing the farnesyl diphosphate synthase gene ispA Farnesyl diphosphate synthase ispA synthesising GPP and FPP significantly contributes to the biosynthetic system of the terpenoid carbon skeleton [38]. In this work, the recombinant E. coli Z25 was constructed via transforming the recombinant plasmid pACYCDuet-1-ispA into E. coli Z20 (Additional file 1: Fig. S1/S11a). The titer of cembratriene-ol in E. coli Z25 was significantly improved compared to that of E. coli Z24 (Fig. 2a). The titer and productivity of cembratriene-ol in E. coli Z25 were also enhanced compared those of E. coli Z24. The overexpression of farnesyl diphosphate synthase ispA significantly enhanced the synthesis of cembratriene-ol in E. coli. Meanwhile, cembratriene-ol was also efficiently synthesised in S. cerevisiae BY-T20 with high production level of geranylgeranyl pyrophosphate (GGPP) via enhancing MVA pathway, the titer of which reached approximately 1.56 mg L − 1 [39]. The glucose consumption of E. coli Z25 was close to that of E. coli Z24, and cell growth was not inhibited compared with E. coli Z24 (Additional file 1: Fig. S11b/c). ispA overexpression has little effect on the glucose consumption and cell growth of recombinant E. coli.

Enhancing the production of cembratriene-ol via optimised fermentation
Besides engineering strategies focus on biosynthetic pathways, the microbial cell factories performance can be greatly enhanced by appropriate optimization of the fermentation conditions [40]. Fermentation processes have been optimised to further improve the production of target products in recombinant E. coli, such as medium optimisation and environmental parameter optimisation. Carbon source and nitrogen source, as important medium components, play crucial roles in the production of primary and secondary metabolites and the cell growth [41]. The production level of cembratriene-ol was further improved by adding different proportions of glucose and yeast extract (Fig. 3). When different proportions of added glucose and yeast extract were 4:2, 4:4, 4:6, 4:8, 4:10, and 4:12, the titers of cembratriene-ol in E. coli Z25 were 9.94, 21.51, 22.47, 30.37, 21.00, and 20.48 mg L − 1 , respectively. When the proportion of added glucose and yeast extract was 4:8, the titer, productivity, and conversion yield of glucose to cembratriene-ol were the highest, increasing by 4.75-, 4.75-, and 2.75-folds that of the control (without addition of glucose and yeast extract), respectively. It was indicated that high nitrogen source concentration promoted production of cembratrieneol. It was also found that the addition of nitrogen source with high concentrations was advantageous for efficient accumulation of rhamnolipids [41,42]. There was residual glucose when glucose and yeast extract were supplemented. When the proportion of added glucose and yeast extract was 4:2, the residual glucose concentration was the highest (up to 2.99 g L − 1 ). High concentrations of glucose cannot be fully utilised compared to yeast extract at high concentrations. The synthesis of primary or secondary metabolites and/or the cell growth can be usually influenced bythe metabolic rate of carbon sources [41]. The growth of recombinant E. coli was significantly improved by the addition of glucose and yeast extract in different proportions compared with that of the control (without addition of glucose and yeast extract). When the proportion of added glucose and yeast extract was 4:8, the cell density was the highest and the cell growth was the fastest. The maximum OD 600 and the maximum specific growth rate were increased by 3.00-and 3.23folds those of the control (without addition of glucose and yeast extract), respectively (Additional file 1: Fig.  S12a/b). However, when the proportion of added glucose and yeast extract was 4:2, the cell growth was lower than those of other proportions of added glucose and yeast extract. It was also found that astaxanthin production in Xanthopyllomyces dendrorhous could be promoted by high C/N, but the cell growth could be inhibited by high glucose [40]. Meanwhile, the pH changes of fermentation broth at different C/N ratios were determined, and the pH values at 4:8, 4:10, and 4:12 (C/N ratios) were 6.55, 6.60, and 6.64 at 48 h, respectively. Nitrogen sources can promote cell growth of microorganisms. Too many nitrogen sources can make the cell row too vigorously and the pH value is too high, which is not conducive to the accumulation of metabolites. Improper C/N ratios also affect the proportion of nutrients absorbed by the cell, directly affecting the cell growth and the product synthesis.
The above results showed that glucose could not be completely utilised by recombinant E. coli. The concentration of residual glucose reached 1.14 g L − 1 at 48 h Student's t-test was used to statistically analyze data, and statistical significance was set at p value < 0.05. Asterisks indicate significant differences compared to E. coli Z25 (***, p < 0.01; **, p < 0.05; *, p > 0.05). p < 0.05 was considered statistically significant when the proportion of added glucose and yeast extract was 4:8. It was presumed that the extension of the fermentation period could increase the production of cembratriene-ol in E. coli Z25. In this study, the fermentation period was optimised to improve cembratrieneol production. As shown in Fig. 3c, the cembratriene-ol production by E. coli Z25 was further enhanced during the extension of fermentation time. The titers of cembratriene-ol reached 42.13 and 53.72 mg L − 1 at 60 and 72 h, respectively (Fig. 3c). The production intensities of cembratriene-ol also increased to 0.70 and 0.75 mg L − 1 h − 1 at 60 and 72 h, respectively (Fig. 3d). The titer of cembratriene-ol at 72 h was 1.8-folds higher than that at 48 h and 3357.5-folds that of the initially constructed E. coli Z14. The conversion yield of glucose to cembratriene-ol reached 6.81 mg g − 1 at 72 h. It was presumed that glucose was gradually converted into cembratriene-ol during extension of fermentation time.

Introduction of the heterologous MVA pathway
E. coli only has the natural DXP pathway, which includes 7 enzymatic steps with an adenosine triphosphate (ATP) and a reducing agent (NADPH) [1]. Intermediate enzymes and their genes from DXP pathway have been identified, and terpenoid units are synthesized from DXP starting from the condensation of glyceraldehyde 3-phosphate and pyruvate in this pathway [43,44]. Other alternative routes (e.g., from pentose) along with original DXP pathway can be utilized to improve several terpenoids production [1]. MVA pathway commonly found in archaea and eukaryotes is another known biosynthetic pathway of terpenoids [1,45]. MVA pathway starting from condensation of 2 acetyl-CoA includes 6 enzymatic steps with 3 ATPs and 2 NADPHs [44]. The heterologous MVA pathway was commonly introduced to improve production of terpenoids in E. coli [1]. In this study, 6 genes (orfA, orfB, orfC, orfD, hmgr, and orfE) encoding enzymes from MVA pathway of Streptomyces were synthesized after codon optimization to introduce a heterologous MVA pathway in E. coli Z25, including acetyl-CoA carboxylase, acetoacetyl-CoA synthase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, and mevalonate diphosphate decarboxylase (Fig. 4a). Genes orfA, orfB, and orfC were overexpressed with pRSFDuet-1 and genes orfD, hmgr, and orfE were overexpressed with pETDuet-1 to construct E. coli Z26 (Additional file 1: Fig. S13a). Genes orfA, orfB, and hmgr were overexpressed with pRSFDuet-1 and genes orfC, orfD, and orfE were overexpressed with pETDuet-1 to construct E. coli Z27 (Additional file 1: Fig. S13a). Production of cembratriene-ol was enhanced by introduction of the heterologous MVA pathway from Streptomyces. The titer of cembratriene-ol in E. coli Z27 was significantly increased by 1.13-folds that of E. coli Z25 (p value < 0.05) at 24 h (Fig. 4b). However, the titer of cembratriene-ol in E. coli Z27 was not significantly improved compared with that of E. coli Z26. Overexpression of key enzyme genes using plasmids with high copy numbers were also preferred for production of terpenoids [1,46]. The conversion yields of glucose to cembratriene-ol in E. coli Z26 and E. coli Z27 were lower than that of E. coli Z25, which were consistent with glucose consumption yields of these strains (Additional file 1: Fig. S13b). Meanwhile, it was also found that with the extension of fermentation time, the titers of cembratriene-ol in E. coli Z26 and E. coli Z27 were not significantly improved compared with that of E. coli Z25 (data not shown). It was presumed that the introduction of the heterologous MVA pathway from Streptomyces might cause a metabolic imbalance in the late fermentation stage. Glucose was not gradually converted into cembratriene-ol in E. coli Z26 and E. coli Z27. However, cell growth of E. coli Z26 and E. coli Z27 was inhibited b Effects of introduction of the heterologous MVA pathway on the production of cembratriene-ol at 24 h. Student's t-test was used to statistically analyze data, and statistical significance was set at p value < 0.05. Asterisks indicate significant differences compared to E. coli Z25 (**, p < 0.05; *, p > 0.05). p < 0.05 was considered statistically significant compared with that of E. coli Z25 (Additional file 1: Fig.  S13c/d). More byproducts were formed in E. coli Z26 and E. coli Z27 than E. coli Z25. For example, the concentrations of lactic acid in E. coli Z26 and E. coli Z27 reached 90 and 69 mg g − 1 (DCW), which were higher than that in E. coli Z25. Meanwhile, the acetic acid concentrations in E. coli Z26 and E. coli Z27 reached 3.15 and 5.15 g g − 1 (DCW), which were higher than that (2.89 g g − 1 (DCW)) in E. coli Z25 (Additional file 1: Fig. S14).

Enhancing the production of cembratriene-ol via overexpressing ispD and ispF and fermentation scale-up in 15-L fermenter
It was found that the titers of cembratriene-ol in E. coli Z26 and E. coli Z27 heterologously overexpressing MVA pathway from Streptomyces were not significantly improved compared with that of the control (E. coli Z25). The enzyme encoded by dxs can condensate glyceraldehyde-3-phosphate with pyruvate to produce deoxyd-xylulose in the DXP pathway, and deoxy-d-xylulose is converted to the C5 isoprene subunits (IPP and DMAPP) by a series of additional isoprenoid enzymes encoded by dxr, ispD, ispE, ispF, ispG, and ispH in subsequent sequential reactions [33]. Enzymatic bottlenecks genes (dxs, idi, ispD, and ispF) reported were overexpressed to increase the flux through the upstream DXP pathway [33,47]. Therefore, key genes ispD and ispF from the DXP pathway were additionally overexpressed to enhance synthesis of intermediate metabolites to improve the accumulation of cembratriene-ol in this study. The genes ispD and ispF were overexpressed using pRSFDuet-1 and pETDuet-1 with high copy numbers in E. coli Z25 to construct the recombinant strain E. coli Z28 (Additional file 1: Table S1). The cembratriene-ol titer in E. coli Z28 reached 83.91 mg L − 1 at 72 h, which was increased by 1.56-folds that of E. coli Z25 (Fig. 5a).
Through fermentation optimization in the 250 mL shaking flask, it was verified that addition of glucose and organic nitrogen source (yeast extract) was conducive to the production of cembratriene-ol and cell growth. However, the concentration of added glucose in the shaking flask was low and not used for high-density fermentation in a 15-L fermenter. In this study, a fed-batch culture with DO-stat was adopted to achieve high-density fermentation in a 15-L fermenter. The feeding medium was fed at a dissolved oxygen (DO) concentration of 45%. Among feeding media, the carbon source was glucose, and the nitrogen source was mainly provided by adding NH 3 H 2 O (inorganic nitrogen source). In order to promote cell growth, a small amount of yeast extract and peptone was added in high concentration glucose fed to supplement a certain amount of organic nitrogen source. Feeding medium included 500 g L − 1 glucose, 7.33 g L − 1 MgSO 4 , 4.0 g L − 1 yeast extract, and 4.0 g L − 1 tryptone. When the cell density OD 600 reached 15 at 8 h, IPTG was added to induce expression of key enzyme genes (e.g. dxs, idi, ispA, ispD, ispF, ggpps, and cbts*), which could be used to efficiently synthesize cembratriene-ol in E. coli Z28.

Fig. 5
Enhancing the production of cembratriene-ol via overexpressing ispD and ispF and fermentation scale-up in 15-L fermenter. a, Enhancing the production of cembratriene-ol via overexpressing ispD and ispF. −, no gene overexpression; +, gene overexpression using pACYCDuet-1; 2+, gene overexpression using pCDFDuet-1; 3+, gene overexpression using pETDuet-1; 4+, gene overexpression using pRSFDuet-1. Student's t-test was used to statistically analyze data, and statistical significance was set at p value < 0.05. Asterisks indicate significant differences compared to E. coli Z25 (**, p < 0.05). p < 0.05 was considered statistically significant. b, Fermention scale-up in 15-L fermentor The titers of cembratriene-ol in 15-L fermenter reached 371.2 mg L − 1 at 80 h, which was significantly enhanced compared with that of the initially constructed E. coli Z14 (Fig. 5b). The highest OD 600 reached 102.3 at 80 h. The residual glucose concentrations were below 0.5 g L − 1 (Fig. 5b). Carbon substrate feeding speed was connected to the DO level. When E. coli cells were actively growing with sufficient glucose, they required a lot of oxygen and the DO level would become very low. Once glucose is depleted, the DO level started to rise. Meanwhile, the titer of cembratriene-ol in the recombinant E. coli overexpressing dxs, idi, and ggpps using different ribosomal binding sites (RBS) combinatorics reached 78.9 mg L − 1 at 120 h in a 50-L fermenter, and the dry cell weight reached 15.3 g L − 1 [12].

Conclusion
The tobacco leaf-derived cembratriene-ol was successfully synthsized in recombinant E. coli. The systematic regulation of ggpps, cbts*, dxs, and idi expression levels could promote the production of cembratriene-ol. The overexpression of the farnesyl diphosphate synthase gene ispA, ispD, and ispF was significantly for enhancement of the cembratriene-ol titer. The cembratriene-ol titer in recombinant E. coli could be importantly improved by fermentation optimisation and introduction of the heterologous MVA pathway from Streptomyces. The cembratriene-ol titer in the 15-L fermenter was the highest titer reported. Systematic optimization has great potential in facilitating high-level production of cembratrieneol production in E. coli.

Strains and plasmids
The strains constructed and used are listed in Additional file 1: Table S1. E. coli JM109 was used for cloning, and E. coli BL21(DE3) was used as the initial strain to produce cembratriene-ol. The plasmids used and constructed are listed in Additional file 1: Table S2. The primers used for gene cloning and plasmid construction are listed in Additional file 1: Table S3.

Detection of cembratriene-ol
After fermentation, 30 mL of fermentation broth with cells was collected from each flask. All flask fermentations were performed in triplicate. Cells in each flask were lysed using a SCIENTZ-IID Ultrasonic Cell Crusher from Ningbo Scientz Biotechnology Co., Ltd. (Ningbo, China). Equal volumes of the extractant (n-hexane:ethyl acetate = 1:1) were added to the mixtures, lysed, mixed for 30 min, and centrifuged at 5000×g and 4 °C for 10 min. The supernatant was collected and dried at 30 °C using a RE100-Pro rotary evaporator (Scilogex, New Hampshire, USA). To reconstitute the dried samples, 3 mL of ethyl acetate was used, and reconstitution was determined using GC-MS. The TraceGOLD TG-5MS GC column (30 m × 0.25 mm × 0.25 µm) of Thermo Fisher Scientific Co., Ltd. (Waltham, USA) was used to determine cembratriene-ol. The column temperature was 100 °C and its hold time was 0 min; it was increased to 250 °C at a rate of 10 °C min -1 and its hold time was 5 min. MS data of samples were recorded at 70 eV (EI) and m/z (rel. intensity in %) as total ion current. Full scan mode (m/z 35-350) was used to collect the data. The productivity of cembratriene-ol was calculated over the whole culture. The conversion yield of glucose to cembratriene-ol was calculated by dividing the cembratriene-ol titer (mg L -1 ) by the glucose consumption concentration (g L -1 ). Three parallel experiments were independently performed, and data are reported as mean ± standard deviation (SD).

Determination of the cell density (OD 600 ) and the specific growth rate
Recombinant E. coli culture broth (1 mL) was used to determine the absorbance at 600 nm (OD 600 ). The specific growth rate plots were drawn from OD measurements using the software OriginPro 8.5.1. The data processing steps are as follows: (a) Fitting of time and OD to obtain data 1; (b) Differential of OD to time to obtain data 2; (c) Data 2 were divided by data 1 to obtain specific growth rates (µ), which included 100 data and the total fermentation time was evenly divided into 100 equal parts. The obtained specific growth rates (µ) including 100 data and time divided into 100 equal parts were used to draw the specific growth rate plots using the software OriginPro 8.5.1. Three parallel experiments were independently performed, and data are reported as mean ± standard deviation (SD).